This application takes advantage of monoclonal antibody 3E10, an unusual lupus-derived auto-antibody that can penetrate human cells without toxicity. 3E10 provides a unique platform for developing bi-specific antibodies and antibody-peptide fusion proteins that can be tailored to target intracellular factors for melanoma therapy. The 3E10 scFv fragment (a recombinant single-chain protein composed of the variable regions of the 3E10 heavy and light chains joined by a short linking domain) can penetrate into cells and nuclei and can deliver protein cargoes through recombinant fusion. We will test the hypothesis that such bi-functional molecules can be rationally designed to specifically target melanoma cancer cells by exploiting two well established vulnerabilities in human melanoma: (1) oncogene addiction (due to activated or over-expressed oncogenes such as MDM2) or (2) synthetic lethality (due to aberrations in DNA repair capacity in the melanoma cells). In preliminary work, we have already demonstrated the ability of 3E10 scFv to penetrate into cancer cell nuclei within human solid tumor xenografts in mice following systemic administration. To achieve our goals, we have assembled a team of investigators (Drs. Glazer, Sweasy and Weisbart) with complementary expertise in cancer therapy, DNA repair, and antibody technology. This proposal also takes advantage of a large collection of primary melanoma cell populations established directly from patient samples by Dr. Ruth Halaban, the PI of the Yale SPORE in Skin Cancer. The SPORE collection includes over 300 melanoma samples, from which over 100 cell cultures have been established. Dr. Halaban will make this collection available for the work of this proposal to test the efficacy of the proposed bi-functional antibodies in the context of clinically relevant human melanoma cultures. In Aim 1, we will target oncogene addiction in human melanomas linked to over-expression of MDM2. A bi-functional antibody with 3E10 scFv combined with the scFv fragment of an antibody to MDM2 (3G5) has already been produced. MDM2 is a negative regulator of p53, and melanomas with over-expression of MDM2 have addiction to this factor. We have preliminary evidence that disrupting MDM2/p53 interactions with the 3E10-3G5 antibody causes apoptosis and inhibits melanoma growth. In Aim 2, we will combine 3E10 scFv with a peptide derived from the DNA repair factor, Partner and Localizer of BRCA2 (PALB2), that we have discovered can disrupt PALB2/RAD51 DNA repair complexes by blocking key interactions between these proteins. This PALB2 peptide is predicted to be synthetically lethal to human cancer cells with abnormalities in either of two DNA repair pathways, base excision repair (BER) or homology-dependent repair (HDR). We expect this work to provide proof-of-principle to establish a novel platform for developing antibody- based reagents to selectively disrupt intracellular pathways, by combining the specificity of antibody-antigen or protein-protein interactions with the unique properties of the cell-penetrating 3E10 scFv fragment.